Surface coatings for hot-melt adhesive film

ABSTRACT

A low temperature melt film such as EVA is prepared for laser capture microdissection by having a thin specimen non-adhering coating in the range of 0.1% to 10% of the total film thickness placed on the sample exposed side of the film. When the film is brought into contact with the specimen, the specimen non-adhering coating prevents non-specific transfer due to sticky adherence of portions of the sample. At the same time, the non-adhering coating on the low temperature melt film surface can stabilize and protect the low temperature melt film against variations in performance due to ambient humidity and temperature variation. Upon appropriate heating for laser capture microdissection, the barrier of the thin coating allows conventional film melting with otherwise uninhibited adhesion of selected cell areas to the film. Coatings on the low temperature melt film (EVA) surface in selected locations is made by applying film-forming material from a volatile solvent-based solution, followed by evaporation of the solvent. The coating solution can be applied by spraying, dipping, or adding exact volumes to a surface with a micropipet. Spreading a measured volume of solution over the surfaces can coat flat surfaces. The coating thickness can be controlled by the volume and concentration of coating solids added to a known area.

This invention relates to laser capture microdissection (LCM). Morespecifically, this disclosure sets forth an improved film for use withlaser capture microdissection in which the film is used in contact withthe specimen and a coating is provided over the film which prevents nonspecific transfer of specimen without inhibiting desired laser capturemicrodissection.

BACKGROUND OF THE INVENTION

Laser capture microdissection (LCM) is known. Specifically, a samplesuch as a tissue specimen has multiple cell areas scattered throughout.Taking the case of a biopsy for cancer, these cell areas can includecancer cells, pre-cancer cells, irritated cells, normal cells and othertissue. The diagnostician usually desires only one type of cell or cellportion from one cell area—say for purposes of this example—the “pure”cancer cells. Further, the diagnostician requires sufficient quantity ofthe cells from the selected cell areas to perform further meaningfuldiagnosis. As a consequence, multiple samples of similar cells from oneor more specimens are frequently required.

Laser capture microdissection is preferably performed with a transparentlow temperature melting film such as (polyethylene/vinyl acetate), CAS24937-78-8) (also known as EVA) manufactured by the Du Pont Corporationof Wilmington, Del. The reader will understand that many othermanufacturers vend similar materials.

Specifically, a specimen—usually on a glass slide is overlaid with thelow temperature melting film such as EVA. Thereafter, a cell area of thesample wanted for dissection is visualized and located, preferablythrough a microscope. When location has occurred, precision heating andmelting of the low temperature film occurs overlying the visualized cellarea. The precision heating of the low melting point film occurs byhaving a laser heat that portion of the low melting point film thatoverlies the cell area targeted for microdissection. At the precisionheated area, flow of the melted EVA occurs from the film to the sampleonto the visualized cell area. The sample is adhered to the melted EVAupon solidification of the precision melted portion of the film.Thereafter, the film is removed from the sample, preferably by placingthe film overlying the specimen under tension and lifting the film awayfrom the sample. Typically, the sample at the adhered identified cellarea sticks to the film with the result that a microdissection occurs.The reader will understand that the film a coating can either bestretched or can reside on a support surface.

In LCM, at least a major part of the mechanism of adhesion ismechanical. The heated film overlying the selected cell area flows intoand around the specimen portions to be microdissected. Thereafter, theheated and flowed low temperature melt film solidifies. When the film iswithdrawn, the physical interference between the flowed and solidifiedfilm material and the cell area of the specimen intended formicrodissection causes the film when it is withdrawn to “pull” theselected cell area from the remainder of the specimen. Microdissectionoccurs.

The primary reason for low temperature in the melting of the film is toavoid damage to or change the nature of the specimen. EVA, amongplastics has a uniquely low melting range, which can be controlled bythe manufacture. Such manufacture control can include the addition of avariety of ingredients (e.g. rubber) to adjust the melting point andother properties. The ethylene part of the polymer can be used forproperty variations.

Understanding this mechanical adhesion, the reader can quicklyunderstand the reason for using a low temperature melt film. It isobviously desired to remove the targeted portion of the specimen forfurther diagnosis. Where the melted film flows in and around thetargeted portion of the specimen, undue heating changes the nature ofthe targeted cellular material and makes may invalidate subsequentanalysis or diagnosis. This subsequent analysis, or diagnosis, includespotentially a variety of methods for research and clinical evaluation,such as genetic, immunological, enzymatic, and protein analysis.

At the same time, precise and precision transfer of the intended cellarea is required. Such precise transfer must gather only the identifiedcell area—say for the sake of the example discussed above—the “pure”cancer cells. In the LCM, recovery of materials from cell areas otherthan cellular material within the identified cell area is referred to as“non-specific transfer.” Non specific transfer can be detrimental tofurther analysis including biological amplification techniques such asPCR. Some applications of LCM are sensitive to very low levels ofundesired sample areas.

To avoid non-specific transfer, LCM as currently practiced has divideditself into two broad techniques. In one technique, known as non-contactLCM, the film to which attachment occurs is held spaced a small butconstant interval from the sample. When local heating of the EVA orother film utilized occurs, the film expands across the spatialinterval, and adheres to the specimen at the visualized portion. Whenthe film is removed, microdissection occurs. A solution to this problemhas been to devise various means of spacing the EVA film away from thetissue, so that the EVA contacts the tissue only in the selected spotsby its expansion during laser melting.

The EVA expands in a column or pedestal at the area of activation byabout 10% to 15%, melts into the tissue sample, and then withdraws awayslightly, retaining a microscopic tissue sample upon cooling. Thedesired spacing of the EVA surface away from the tissue sample is of theorder of 10 micrometers. There are various ways of providing thespacing, including putting a spacer film on the EVA, located so thespacer film is in contact with an area of the tissue away from thedesired sampling point and holding the EVA surface away from the tissue.With regard to such spacing techniques, non specific transfer may alsooccur even though a spatial interval is present, due to loose or weaklyadhering substances and unevenness of tissue.

It is desirable to press the surface of the EVA against the tissuesample with controlled force, as one of the control parameters of tissuesampling, with the spacing preventing actual contact of the EVA andtissue. The coating of this disclosure may be desirable to prevent nonspecific transfer in this method also.

It is to be understood that non-contact microdissection is not withoutproblems. Specifically, maintaining the film at a precise closely spacedinterval from the specimen at the visualized cell area is difficult.Precision control of the parameters of contact of the low temperaturemelt film to the sample is difficult.

In another technique of LCM, which is directly applicable to thisinvention, the film is brought into direct contact with the specimenbefore melting occurs. In the past, this direct contact with thespecimen has caused non-specific transfer. Specifically, the filmused—usually EVA—is naturally tacky. This natural tackiness results fromthe softening point of the film that is required to minimize damage tothe microdissected cell area being removed.

Complicating LCM, the biological specimen is also non-homogenous. Thespecimen typically contains proteins, carbohydrates, fats oils and othercellular materials in an irregular matrix. Portions of this irregularmatrix can preferentially adhere to tacky surfaces of the film withoutthe laser heating. Thus, when the targeted material is adhered in theLCM process and withdrawn from the specimen, undesired adjacent cellareas of the specimen are removed and transported by the film. It isthis non-specific transfer which it is the purpose of this invention toavoid.

SUMMARY OF THE INVENTION

A low temperature melt film such as EVA is prepared for laser capturemicrodissection by having a thin specimen non-adhering coating which ishard and non tacky. The hard and non-tacky coating may be in the rangeof 0.1% to 10% of the total film thickness placed on the sample exposedside of the film. Alternately, a coating may be added to EVA in therange of two to two hundred micro inches. The coating may be applied byany acceptable method including solvent based coatings, laminations andthe like. When the coated film is brought into contact with thespecimen, the specimen non-adhering coating prevents non-specifictransfer due to sticky adherence of portions of the sample. Further, thehard coating will allow use of techniques to remove non-specifictransferred material, such as by brushing or washing away attractedmaterial from the hard coating. At the same time, the non-specimenadhering coating on the low temperature melt film surface can stabilizeand protect the low temperature melt film against variations inperformance due to ambient humidity and temperature variation. Uponappropriate heating for laser capture microdissection, the barrier ofthe thin coating allows conventional film melting with otherwiseuninhibited adhesion of selected cell areas to the film. Coatings on thelow temperature melt film (EVA) surfaces in selected locations are made,for example by applying film-forming material from a volatilesolvent-based solution, followed by evaporation of the solvent. Thecoating solution can be applied by spraying, dipping, or adding exactvolumes to a surface with a micropipet. Spreading a measured volume ofsolution over the surfaces can coat surfaces. The volume can control thecoating thickness and concentration of coating solids added to a knownarea.

One of the simplest and most practical ways of making a coating on theEVA surface in selected locations is by applying an appropriatefilm-forming material from a volatile solvent-based solution followed byevaporation of the solvent. A water or water-ethanol solution is optimalbecause it does not attack and deform the EVA surface. The usual organicsolvent solutions of a polymer are unsuitable because the solventinteracts with the EVA and spoils its surface.

Three materials have been found suitable for this application, althoughother candidates for water or ethanol solution film formers arepossible, including variations of these materials. The EVA used isDuPont Elvax 410. The film materials used are polyvinyl alcohol (Mowiol40-88 Hoechst, mw 127,000, Aldrich Chemical Co. 32,459-0, CAS9002-29-5), polyvinylpyrrolidone (PVP, Fisher Scientific Co. BP431-100,mw 40,000, CAS 9003-39-8), and chitosan (Aldrich Chemical Co. 44,887-7,medium mw, CAS 9012-76-4). These are used in solution in water or 50%ethanol in water at concentrations from 0.1 g/100 ml to 10 g/100 ml,most commonly 1 g/100 ml. The chitosan solutions contain 5% by volumeacetic acid in the water to solubilize the chitosan.

To apply the coatings evenly it may be necessary to prepare the surfaceof the EVA by plasma etching to make it solution wettable. Also, somecoating materials, particularly PVP, require etching of EVA to givesatisfactory film adhesion. Exposure for about one minute to a plasma inair at a pressure of approximately one-half torr is sufficient. Thearticles to be treated are placed in a six-inch diameter glass vacuumdesiccator with internal electrodes, connected to a vacuum leak-testingprobe as a high-voltage high-frequency current source.

The coating solution can be applied by spraying, dipping, or addingexact volumes to a surface with a micropipet. Applying a measured volumeof solution of known concentration over known surface area, as on caps,can control the film thickness added to the known area.

It has also been proposed to accomplish laser capture microdissectionutilizing a conical surface. In this application, successive applicationand rotation of a conical surface enables a high concentration ofsimilar cells at closely spaced intervals over the flat surface.Utilizing this disclosure, rod shapes can be coated completely.

It has also been proposed to provide such rods with spacer bands. Thesespacer bands contact the specimen or slide surface and hold the lowtemperature melt film a given distance away from the specimen. Suchspacer bands can be protected against non-specific transfer with thecoatings set forth herein. Dipping, with rotation to improve evenness ofcoating application may be used to evenly coat the spacer bands. Rodscan also be coated by addition of solution from a micropipet duringrotation.

Pattern coating, or control of the area coated by a film can be done bymasking during the plasma etching process so that the non etched surfaceof EVA is not wetted by the solution. Various methods of printingtransfer of solution to the surface can also be used to apply thesolutions to coat the EVA in selected regions. Addition of ethanol tothe solution of coating material will vary the wettability of thesolution on the EVA.

The thickness of the film can be estimated by calculation from thevolume and concentration added to a known area. The thickness of thefilm can also be measured microscopically by counting interferencefringes with the film against a glass slide surface or by measuring thefocus location change when focusing on the upper and lower surface of afilm. Inclusion of a dye, preferably fluorescent, in the film can beused to calibrate the thickness by color intensity measurement. A dyealso makes it easy to see the location of the film, which wouldotherwise be difficult. An example dye is rhodamine B (CAS 81-88-9) in aconcentration of 100 micrograms/ml in the solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of conventional laser capture microdissectionillustrating a slide mounted specimen covered with a low temperaturemelt film having a cell area visualized for microdissection andillustrating schematically the laser path for heating and adhering thefilm to the cell area of the specimen;

FIG. 2 is an enlarged cross section illustrating the sample, the film,and the coating between the film and sample, a portion of the film shownmelted and expanding through the coating to form normal laser capturemicrodissection;

FIG. 3 is a further enlarged section of the film illustrating the moltenportion of the film breaking through coating of this invention andadhering to the specimen at least through some mechanical attachment;

FIG. 4 illustrates in side elevation section the coating of thisinvention applied to a cylindrical surface;

FIG. 5 illustrates in side elevation section the coating of thisinvention applied to a spacer band; and,

FIG. 6 illustrates a pattern prepared surface coated in a pattern withthe coating of this invention, the pattern prepared surface being idealfor flat films, especially tapes and the like.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Referring to FIG. 1, eye E observes through microscope M slide S havingspecimen P. Schematically shown on slide S at specimen P is two types oftarget cell areas, 14-15. Those have familiarity with some biopsies willunderstand that cells are found in a matrix in such samples. Here weschematically illustrate the cells of interest as target cell area 15.The reader will understand that eye E is schematic; present practiceincludes the use of digital displays and the like.

We schematically show a microscope configuration. More particularly, weillustrate schematically laser L deflected by mirror 20 upward throughslide S and specimen P.

Most importantly, low temperature melt film F immediately overliesspecimen P. It is this film which is locally and precision heated bylaser L. When such heating occurs, precision melting of low temperaturemelt film F occurs and microdissection of specimen P at target cell area15 can occur. Sequentially, light from laser L couples to lowtemperature melt film F and causes melting. Thereafter, molten lowtemperature melt film F attaches to specimen P. When the film isremoved, the attached target cell area 15 is removed with the film.

Observing FIG. 2 taken along section lines 2—2 of FIG. 1, a sideelevation view is taken at the sample. It is clearly seen that the filmis in contact with the sample. There has been an effort to schematicallyrepresent the irregular top 18 of specimen P.

Turning to FIG. 3, a further enlarged view of the LCM is shown. Lowtemperature melt film F is shown having the non-adhering coating C ofthis invention attached. Beam 20 from laser L is shown passing upwardthrough slide S, specimen P, and coupling to low temperature melt filmF. As a result, molten column 25 passes through non-adhering coating Cand allows molten columns 25 to adhere to specimen P at cell area 15.When low temperature melt film F is raised from specimen P,microdissection occurs.

We have already given the composition of non-adhering coating C on lowtemperature melt film F. Some attention can be directed here to thethickness of non-adhering coating C. Typically, and on a broad range,non-adhering coating C is between 0.1% and 5% of the total thickness oflow temperature melt film F. In a narrow range, such thickness is from3% to 0.5% with optimum thickness being the range of 1%.

The reader will also understand that the coating can be limited in termsof coating thickness. Such coating thickness can nominally be 1 micronin a narrow range, 0.5 to 5 microns in an intermediate range, and 0.1 to10 microns in a broad range. Given that fact that films may vary widelyin total thickness, the limitations on coating thickness are preferred.

Low temperature melt film F nominally is in the range of 100 micronsthick, although total thickness of the film may vary. It is to beunderstood that low temperature melt film F is capable of transmittingall (tensile) forces necessary for the microdissection. Non-adheringcoating C does not appreciably contribute to such tensile forces.

In one embodiment of this invention, non-adhering coating C is ofchitosan and as a consequence very brittle. A coating which breaks awaysharply from the EVA wetted LCM spot is important, so that a sharp edgeor departure zone is maintained between the film heated to becomeadhesive and the surrounding non-activated film which is coated againstthe non-specific transfer. All that is required is that non-adheringcoating C is sufficiently non-adhering to specimen P so thatnon-specific transfer does not occur.

In what follows are specific examples of film and film coating, which wehave used:

Exam- EVA ple Coating Eva Film Solution Treatment Thickness 1polvinylpyrrolidone 1% polyvinyl- 50% ethanol plasma etch 0.3micrometers (PVP, Fisher Scientific Co. pyrrolidone, in water BP431-100,mw 40,000, CAS 9003-39-8)\on DuPont Elvax 410 2 polyvinylpyrrolidone 1%polyvinyl- plasma etch 0.3 micrometers (PVP, Fisher Scientific Co.pyrrolidone BP431-100, mw 40,000, CAS in water 9003-39-8)\on DuPontElvax 410 3 polyvinyl alcohol 5% Mowiol, 50% ethanol 10 micrometers(Mowiol 40-88 Hoechst, mw in water 127,000, Aldrich Chemical Co. 32)459-0, CAS 9002-89- 5) on DuPont Elvax 410 4 chitosan 1% chitosan 5%acetic acid, plasma etch 0.1 micrometer (Aldrich Chemical Co. 50%ethanol 44,886-7, medium mw, CAS in water 9012-76-4) on DuPont Elvax 410

We have shown the coating here applied to a film surface. It will bejust as apparent that application could as well be to a cylindricalsurface such as conical surface 30 on rod R. In this case, lowtemperature melt film F′ is applied around rod R of FIG. 4 havingnon-adhering coating C′.

With respect to FIG. 5, non-adhering coating C is used on conicalsurface 30 having rimmed 33. Rim 33 with non-adhering coating C thereonprevents non-specific transfer.

Finally, and with respect to FIG. 5, low temperature melt film F hasbeen exposed to plasma at band B. Such exposure enables band B to bewettable. When wetted with the solutions set forth in the examplesabove, non-adhering coating C appears as a band about conical surface30.

The reader will understand that the foregoing specification isexemplary. We show a single coating. The coating could be a laminate, oreven multiple coatings. Further, from what has been previouslyaddressed, the most important aspect of this invention is the use of thehard coating over the convention film, such as EVA. The method ormaterial used for the coating may widely vary.

What is claimed is:
 1. In the process of laser capture microdissectionincluding: providing a specimen having a matrix of cells forexamination; providing a low temperature melt film for laser capturemicrodissection; overlaying and contacting the specimen with the lowtemperature melt film; visualizing the specimen to determine a cell areain the matrix of cells for microdissection; heating the film with alaser overlying the cell area to cause the film to melt and locallyadhered to the cell area; removing the film from the specimen to cause alocally adhered cell area to be removed with the film; the improvementcomprising the step of: placing a specimen non adhering coating on thelow temperature melt film, the specimen non adhering coating exposed toand in contact with the sample, the coating being between 0.1% and 5% ofthe total thickness of the film whereby when the heating of the filmoverlying the cell area causes melting of the film to occur, meltingoccurs through the specimen non adhering coating.
 2. In the process oflaser capture microdissection according to claim 1 and where theimprovement includes: the coating being between 0.1 microns and 10microns thick.
 3. In the process of laser capture microdissectionaccording to claim 1 and wherein the improvement includes: the placedspecimen non adhering coating is selected from the group consisting ofpolyvinyl alcohol, polyvinylpyrrolidone, and chitosan.
 4. In the processof laser capture microdissection according to claim 1 and where theimprovement includes: the placed specimen non adhering coating is in asolution.
 5. In the process of laser capture microdissection accordingto claim 4 and where in the solution comprises water and ethanol andconcentration from the 0.1-10 ge/100 ml.
 6. In the process of lasercapture microdissection according to claim 4 and wherein the improvementincludes: the water and ethanol solution includes 50:50 ethanol: water.7. In the process of laser capture microdissection according to claim 2and where the improvement includes: treating the low temperature meltingpoint film by plasma etching to permit wetting of the film prior toapplying the coating.